Sound is detected and encoded by hair cells and neurons in the inner ear and processed by complex circuits in the central auditory system. Millions of people experience a degree of hearing impairment in either the detection or perception of sound, ranging from profound deafness to tinnitus and learning disabilities. A major objective in hearing research is to find the genes required for hearing in order to improve the diagnosis and treatment of this wide array of peripheral and central auditory disorders. Genetic screens in model organisms, together with positional cloning of human deafness loci, have uncovered many deafness genes and have greatly advanced our understanding of how the auditory system works. However, this knowledge remains incomplete, as highlighted by the fact that the mechanotransduction channel has yet to be identified. Moreover, little is known about the prevalence or etiology of central auditory processing disorders, due to inadequate diagnostic tools and a lack of knowledge of normal auditory circuit assembly and function. The long-term goal of this study is to develop a new method of gene discovery that will complement ongoing screens for deafness genes and expand our understanding of the molecular basis of hearing. We propose to create a new method for rapidly disrupting auditory gene function in vivo. This technique will use Cre-lox technology and RNA interference (RNAi) in the mouse to disrupt the activity of putative deafness genes in restricted cell populations of the inner ear. shRNA production will be linked to activation of a fluorescent marker, permitting easy visualization of neuronal morphology down to the level of the synapse. The method relies on a transgene that carries two sets of incompatible Cre recognition sites, a U6 promoter, a CAG promoter, and the DsRed and Venus coding sequences. These elements are configured such that Cre-mediated recombination results in expression of a gene-specific shRNA and a simultaneous switch from red to yellow fluorescence. The transgene will be targeted to a defined locus in embryonic stem cells, which will be used to establish lines of RNAi mice. These mice can then be crossed to inner ear-specific Cre drivers, circumventing pluripotent effects and lethality. The first aim is to create a Cre-RNAi vector that works effectively in vitro. The second aim is to validate the compatibility of this vector with an in vivo screen in the auditory system by targeting a known deafness gene, GATA3, which is mutated in HDR syndrome. To this end, we will compare the phenotypes of Gata3-RNAi and conventional conditional Gata3 knockout mice, taking advantage of the Venus fluorescence in knockdown mice to visualize changes in cochlear wiring. As well as providing a novel tool for finding genes required for hearing, this technique can be expanded to generate a resource of targeted ES cells that can be screened for function in any region of the nervous system. PUBLIC HEALTH RELEVANCE: Millions of people experience some form of hearing impairment, from profound deafness to central auditory processing disorders that contribute to tinnitus and learning disabilities. The identification of genes necessary for hearing will improve the diagnosis and treatment of a wide array of disorders. The goal of this project is to develop a new method of probing gene function in mice that will advance our understanding of how the auditory system normally functions and what happens when it doesn't in humans.